Influence of the brain perivascular environment on the blood-brain barrier dysfunction during a transient ischemia

Ces dernières années, alors qu’aucun agent neuroprotecteur n’a été efficace en clinique pour parer les dommages de l’ischémie cérébrale, le concept d’unité neurovasculaire (UNV) est apparu comme un nouveau paradigme pour l’investigation et le traitement des accidents vasculaires cérébraux ischémiques. La rupture de la barrière hémato-encéphalique (BHE) localisée au niveau des capillaires cérébraux, et ses corollaires l’œdème vasogénique et l’hémorragie intracérébrale, constituent des événements critiques de la maladie, et restreignent considérablement l’éligibilité des patients à la thrombolyse au rtPA, seul traitement de phase aiguë disponible actuellement en clinique. La complexité des intercommunications qui s’exercent au sein de l’UNV rend difficile l’appréhension de la dysfonction microvasculaire in vivo, soulignant l’importance des études in vitro pour compléter les connaissances dans ce domaine. C’est par cette approche combinée que les travaux effectués au cours de ce doctorat démontrent l’impact de la nécrose cérébrale sur la cinétique de la perte d’intégrité de la BHE au décours de la reperfusion. Cependant, même si l’endothélium microvasculaire demeure fonctionnel après un épisode ischémique dans un contexte non lésionel, il devient vulnérable à certaines molécules comme le rtPA dans une situation de thrombolyse. Ces résultats illustrent le rôle déterminant de l’environnement moléculaire périvasculaire sur la dysfonction de la BHE lors de l’ischémie cérébrale, et orientent les nouvelles stratégies thérapeutiques vers des approches ciblant la protection de l’ensemble de l’UNV.In the recent years, while no neuroprotective agent was clinically effective in reducing brain ischemic damage, the neurovascular unit (NVU) concept emerged as a new paradigm for stroke investigation and treatment. The breakdown of the blood-brain barrier (BBB), localized in brain capillaries, with ensuing vasogenic edema and intracerebral hemorrhage, appears as a critical event of this disease, and severely restricts the eligibility of patients for rtPA thrombolysis, the only acute-phase treatment currently available. The complex intercommunications occurring within the NVU makes the microvascular dysfunction difficult to study in vivo, highlighting the importance of in vitro approaches to complete the knowledge in this field. In this context, the work done in this PhD demonstrates that brain tissue necrosis influences the kinetics of the loss of BBB integrity during reperfusion. However, even when the BBB remains functional in a non-lesional ischemic context, it becomes vulnerable to certain molecules such as rtPA in a thrombolysis situation. These results illustrate the key role of molecular perivascular environment on the BBB dysfunction during cerebral ischemia, and orientate new therapeutic strategies towards the protection of the entire NVU

Influence of the brain perivascular environment on the blood-brain barrier dysfunction during a transient ischemia

Ces dernières années, alors qu’aucun agent neuroprotecteur n’a été efficace en clinique pour parer les dommages de l’ischémie cérébrale, le concept d’unité neurovasculaire (UNV) est apparu comme un nouveau paradigme pour l’investigation et le traitement des accidents vasculaires cérébraux ischémiques. La rupture de la barrière hémato-encéphalique (BHE) localisée au niveau des capillaires cérébraux, et ses corollaires l’œdème vasogénique et l’hémorragie intracérébrale, constituent des événements critiques de la maladie, et restreignent considérablement l’éligibilité des patients à la thrombolyse au rtPA, seul traitement de phase aiguë disponible actuellement en clinique. La complexité des intercommunications qui s’exercent au sein de l’UNV rend difficile l’appréhension de la dysfonction microvasculaire in vivo, soulignant l’importance des études in vitro pour compléter les connaissances dans ce domaine. C’est par cette approche combinée que les travaux effectués au cours de ce doctorat démontrent l’impact de la nécrose cérébrale sur la cinétique de la perte d’intégrité de la BHE au décours de la reperfusion. Cependant, même si l’endothélium microvasculaire demeure fonctionnel après un épisode ischémique dans un contexte non lésionel, il devient vulnérable à certaines molécules comme le rtPA dans une situation de thrombolyse. Ces résultats illustrent le rôle déterminant de l’environnement moléculaire périvasculaire sur la dysfonction de la BHE lors de l’ischémie cérébrale, et orientent les nouvelles stratégies thérapeutiques vers des approches ciblant la protection de l’ensemble de l’UNV.In the recent years, while no neuroprotective agent was clinically effective in reducing brain ischemic damage, the neurovascular unit (NVU) concept emerged as a new paradigm for stroke investigation and treatment. The breakdown of the blood-brain barrier (BBB), localized in brain capillaries, with ensuing vasogenic edema and intracerebral hemorrhage, appears as a critical event of this disease, and severely restricts the eligibility of patients for rtPA thrombolysis, the only acute-phase treatment currently available. The complex intercommunications occurring within the NVU makes the microvascular dysfunction difficult to study in vivo, highlighting the importance of in vitro approaches to complete the knowledge in this field. In this context, the work done in this PhD demonstrates that brain tissue necrosis influences the kinetics of the loss of BBB integrity during reperfusion. However, even when the BBB remains functional in a non-lesional ischemic context, it becomes vulnerable to certain molecules such as rtPA in a thrombolysis situation. These results illustrate the key role of molecular perivascular environment on the BBB dysfunction during cerebral ischemia, and orientate new therapeutic strategies towards the protection of the entire NVU

La barrière hémato-encéphalique lors de l’ischémie cérébrale : une cible thérapeutique

Depuis la preuve de son existence et de son rôle protecteur cérébral, la barrière hémato-encéphalique (BHE), caractérisée par la perméabilité restreinte des cellules endothéliales des capillaires cérébraux, représente un obstacle pour 95 % des futurs médicaments à visée centrale. À l’heure actuelle, une dysfonction de la BHE est trouvée dans un nombre croissant de pathologies telles que les accidents vasculaires cérébraux ischémiques, dont la seule thérapie, une thrombolyse pharmacologique, est limitée à quelques pour cent des patients admis, à cause des effets toxiques des thrombolytiques. Et depuis l’échec clinique de composés neuroprotecteurs prometteurs, de nombreuses études sur l’ischémie cérébrale ont été menées, avec des approches physiopathologiques ou pharmacologiques recentrées sur la BHE dont la complexité structurale s’est élargie à l’ensemble des cellules périvasculaires qui forment une unité fonctionnelle appelée unité neurovasculaire (UNV). Et pourtant, malgré l’identification de nombreux mécanismes moléculaires, le processus de dysfonction de la BHE au décours de l’ischémie/reperfusion demeure insuffisamment décrypté à l’heure actuelle pour expliquer l’action pléiotrope de nouveaux composés pharmacologiques qui pourraient protéger toute l’UNV et représenter de nouveaux traitements

Accumulation of plastid lipid‐associated proteins (fibrillin/CDSP34) upon oxidative stress, ageing and biotic stress in Solanaceae and in response to drought in other species

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Transient oxygen–glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation

International audienceThrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective

Dynamics of the localization of the plastid terminal oxidase PTOX inside the chloroplast

International audienceThe plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOX). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration and setting the redox poise for cyclic electron transport. PTOX activity has been previously shown to depend on its localization at the thylakoid membrane. Here we investigated the dynamics of PTOX localization in dependence on the proton motive force. Infiltrating illuminated leaves with uncouplers led to a partial dissociation of PTOX from the thylakoid membrane. In vitro reconstitution experiments showed that the attachment of purified recombinant MBP-OsPTOX to liposomes and isolated thylakoid membranes was strongest at slightly alkaline pH values in the presence of lower millimolar concentrations of KCl or MgCl2. In A. thaliana overexpressing GFP-PTOX, confocal microscopy images showed that PTOX formed distinct spots in chloroplasts of dark-adapted or uncoupler-treated leaves while the protein was more equally distributed in a network-like structure in the light. We propose a dynamic PTOX association with the thylakoid membrane depending on the presence of a proton motive force

Protein kinase C restricts transport of carnitine by amino acid transporter ATB0,+ apically localized in the blood–brain barrier

International audienceCarnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated

In vitro discrimination of the role of LRP1 at the BBB cellular level: Focus on brain capillary endothelial cells and brain pericytes

International audienceSeveral studies have demonstrated that the blood-brain barrier (BBB) (dynamic cellular complex composed by brain capillary endothelial cells (BCECs) and surrounded by astrocytic end feet and pericytes) regulates the exchanges of amyloid β (Aβ) peptide between the blood and the brain. Deregulation of these exchanges seems to be a key trigger for the brain accumulation of Aβ peptide observed in Alzheimer's disease (AD). Whereas the involvement of receptor for advanced glycation end-products in Aβ peptide transcytosis has been demonstrated in our laboratory, low-density lipoprotein receptor's role at the cellular level needs to be clarified. For this, we used an in vitro BBB model that consists of a co-culture of bovine BCECs and rat glial cells. This model has already been used to characterize low-density lipoprotein receptor-related peptide (LRP)'s involvement in the transcytosis of molecules such as tPA and angiopep-2. Our results suggest that Aβ peptide efflux across the BCEC monolayer involves a transcellular transport. However, the experiments with RAP discard an involvement of LRP family members at BCECs level. In contrast, our results show a strong transcriptional expression of LRP1 in pericytes and suggest its implication in Aβ endocytosis. Moreover, the observations of pericytes contraction and local downregulation of LRP1 in response to Aβ treatment opens up perspectives for studying this cell type with respect to Aβ peptide metabolism and AD

Bexarotene Promotes Cholesterol Efflux and Restricts Apical-to-Basolateral Transport of Amyloid-β Peptides in an In Vitro Model of the Human Blood-Brain Barrier

International audienceOne of the prime features of Alzheimer's disease (AD) is the excessive accumulation of amyloid-β (Aβ) peptides in the brain. Several recent studies suggest that this phenomenon results from the dysregulation of cholesterol homeostasis in the brain and impaired bidirectional Aβ exchange between blood and brain. These mechanisms appear to be closely related and are controlled by the blood-brain barrier (BBB) at the brain microvessel level. In animal models of AD, the anticancer drug bexarotene (a retinoid X receptor agonist) has been found to restore cognitive functions and decrease the brain amyloid burden by regulating cholesterol homeostasis. However, the drug's therapeutic effect is subject to debate and the exact mechanism of action has not been characterized. Therefore, the objective of this present study was to determine bexarotene's effects on the BBB. Using an in vitro model of the human BBB, we investigated the drug's effects on cholesterol exchange between abluminal and luminal compartments and the apical-to-basolateral transport of Aβ peptides across the BBB. Our results demonstrated that bexarotene induces the expression of ABCA1 but not ApoE. This upregulation correlates with an increase in ApoE2-, ApoE4-, ApoA-I-, and HDL-mediated cholesterol efflux. Regarding the transport of Aβ peptides, bexarotene increases the expression of ABCB1, which in turn decreases Aβ apical-to-basolateral transport. Our results showed that bexarotene not only promotes the cholesterol exchange between the brain and the blood but also decreases the influx of Aβ peptides across BBB, suggesting that bexarotene is a promising drug candidate for the treatment of AD

Stroke-Induced Brain Parenchymal Injury Drives Blood–Brain Barrier Early Leakage Kinetics: A Combined in Vivo / in Vitro Study

International audienceThe disappointing clinical outcomes of neuroprotectants challenge the relevance of preclinical stroke models and data in defining early cerebrovascular events as potential therapeutic targets. The kinetics of blood–brain barrier (BBB) leakage after reperfusion and the link with parenchymal lesion remain debated. By using in vivo and in vitro approaches, we conducted a kinetic analysis of BBB dysfunction during early reperfusion. After 60 minutes of middle cerebral artery occlusion followed by reperfusion times up to 24 hours in mice, a non-invasive magnetic resonance imaging method, through an original sequence of diffusion-weighted imaging, determined brain water mobility in microvascular compartments (D*) apart from parenchymal compartments (apparent diffusion coefficient). An increase in D* found at 4 hours post reperfusion concurred with the onset of both Evans blue/Dextran extravasations and in vitro BBB opening under oxygen-glucose deprivation and reoxygenation (R). The BBB leakage coincided with an emerging cell death in brain tissue as well as in activated glial cells in vitro. The co-culture of BBB endothelial and glial cells evidenced a recovery of endothelium tightness when glial cells were absent or non-injured during R. Preserving the ischemic brain parenchymal cells within 4 hours of reperfusion may improve therapeutic strategies for cerebrovascular protection against stroke